PROJECT SUMMARY The long-term aim of our research is to understand the functional organization of the animal cell nucleus. Specifically, we will focus on the non-chromosomal components of the nucleus, collectively referred to as nuclear bodies. Our primary experimental material will be Drosophila, which permits unprecedented genetic, molecular, and developmental analysis. We will also study Xenopus oocytes, where the enormous size of the nucleus and its nuclear bodies make it an ideal system for molecular experiments. Finally, we will examine the biogenesis of an unusual nuclear body in oocytes of the mouse. We will concentrate on four nuclear bodies: the Cajal body (CB), the histone locus body (HLB), speckles (interchromatin granule clusters) and the nucleolus. The function of the nucleolus in ribosomal RNA transcription and ribosome biogenesis is well understood, and there is increasing evidence that components of the pre-mRNA and pre-ribosomal RNA processing machinery are assembled or modified in CBs and HLBs. Some or all of the splicing machinery is stored in speckles before transfer to active transcription sites on the chromosomes. In Drosophila we will concentrate on the CB and its signature protein coilin. We recently identified Drosophila coilin and produced null mutants for coil, the gene that encodes coilin. CBs are not detectable in most cells of the mutants, permitting us to study molecular events in cells that lack both coilin and defined CBs. Specifically, we will examine splicing snRNAs, whose post-transcriptional modifications are thought to occur normally in CBs. We will carry out microarray experiments on ovary and testis RNA to determine which genes are up or down regulated in the absence of coilin. We will also examine the composition and behavior of previously unknown nuclear bodies that are inducible in the Drosophila oocyte under experimental conditions. In Xenopus we will follow the biogenesis of CBs and HLBs throughout the entire period of oogenesis to determine the relationship between these two similar nuclear bodies. We will also examine the prominent population of speckles in the oocyte nucleus to determine whether they associate with specific sites on the giant lampbrush chromosomes, and if so, whether the molecular sequences at these sites can be identified using information from the Xenopus tropicalis Genome Project. Finally, we will examine the ultrastructure and molecular composition of the unusual nucleolus-like bodies that occur in late-stage mouse oocytes. These studies on nuclear bodies in three diverse systems will provide insight into the structure and function of the major nuclear organelles and how they interact during transcription and processing of nuclear RNA.